不同取卵时间对兔ICSI胚胎体外发育的影响

目的探讨不同取卵时间对兔ICSI胚胎体外发育的影响。方法采用Piezo操作系统对实验兔进行辅助体外受精。结果hCG注射后14、16、18h取卵,ICSI后的受精率分别为82.2％、75.9％和0.0％,对受精卵进行体外发育培养,桑椹胚的发育率分别为72.9％、70.0％、0.0％,囊胚的发育率分别为62.2％、53.3％、0.0％。14h和16h之间受精率、桑椹胚率、囊胚率差异不显著（P〉0.05）,但是14h采卵比16h要好;18h和14h、16h之间差异显著（P〈0.05）。结论不同取卵时间影响实验兔的ICSI体外受精率及胚胎的体外发育率,hCG注射后14h取卵最有利于兔ICSI胚胎的发育。     

实验兔胞内单精子注射技术

目的 建立实验兔胞内单精子注射技术(intracytoplasmic sperm injection,ICSI).方法 实验1比较了hCG注射后不同取卵时间对ICSI胚体外发育的影响.实验 2 比较了不同的激活方式对ICSI胚体外发育的影响.实验 3 比较了不同状态的兔精子ICSI胚胎体外发育结果.结果 (1)hCG注射后14 h取卵,其卵裂率、桑椹胚率和囊胚率(82.2%、72.9%和62.2%)都比16 h(75.9%、70.0%和53.3%)的高,但是差异无显著性(P>0.05);18 h取的卵注射后不能卵裂.(2)机械刺激组和离子霉素 6-DMAP组,ICSI后其卵裂率分别为82.2%和81.1%(P>0.05),桑椹胚率分别为72.9%和66.2%(P>0.05),囊胚率分别为51.3%和62.3%(P<0.05),机械刺激组和离子霉素组之间卵裂率和桑椹胚率差异无显著性,但是囊胚率差异有显著性.(3)新鲜精子组和冻融活精子组卵裂率(81.1%和68.8%)和囊胚率(62.3%和40.4%)差异有显著性(P<0.05),而桑椹胚率(66.2%和61.9%)差异无显著性(P>0.05).结论 精子冷冻前后,通过ICSI所得的桑椹胚均能孵化,表明已初步建立了实验兔的ICSI技术.     

猪体细胞核移植重构胚的体外发育

以卵丘细胞为核供体细胞组成重构胚,卵裂率达到56．7％,发育至桑椹胚率达到11．7％,囊胚率为6．7％,显著高于成纤维细胞重构胚（P＜0．05）。本文还研究了卵母细胞的采集方法、激活程序和卵龄对卵丘细胞核移植重构胚体外发育的影响。以血清饥饿法将卵丘细胞诱导G0/G1期,抽吸法／解剖法采集卵母细胞,体外培养33－44h,将卵丘细胞放至去核卵母细胞的卵周隙中,重构胚以钙离子载体A23817或电脉冲结合6－DMAP激活处理,体外培养6d。研究表明,卵母细胞采集方法、激活液中细胞松驰素（CB）、激活程度并不影响重构胚的发育（以卵龄44h的卵母细胞为受体）;而以电脉冲结合6－DMAP激活处理能提高重构胚发育能力（以卵龄33h的卵母细胞为受体）（P＜0．05）。本研究显示,以电脉冲结合6－DMAP激活卵丘细胞重构胚,体外能发育至囊胚。     

兔受精卵显微注射外源基因后体外培养的卵裂发育

从超数排卵的１４只母兔获得４３８枚受精卵,卵龄１６～２２小时．显微操作在带微分干涉和相差的Ｎｉｋｏｎ倒置显微镜下进行．注射针的尖端外也０．５μｍ,离尖端４０和８０μｍ处的外径分别为４．２和６．５μｍ．注射用外源基因是绵羊生长激素基因与ＭＴ－１启动基因藕连的线状ＤＮＡ溶液（１ｎｇ／μｌ）．１４０枚注射的受精卵和未注射的１４５枚受精卵（对照）,在Ｈａｍ’ｓＦ—１０培养液（补充生长因子）中培养（３８℃,５％的ＣＯ２）．结果,培养４８小时后,注射组卵裂发育率分别是：未卵裂７．９％（１１／１４０）、卵裂至２～４细胞期１１．０％（１６／１４０）、卵裂至８～１６细胞期８０．７％（１１３／１４０）．对照组相应的卵裂率分别是４．１％（６／１４５）、１２．４％（１８／１４５）和８３．４％（１２１／１４５）．两组卵裂发育率相近．本实验的显微操作对注射后卵的发育没有产生明显的伤害影响．     

影响猪体细胞核移植重构胚体外发育的若干因素

以卵丘细胞为核供体细胞组成重构胚,卵裂率达到56．7％,发育至桑椹胚达11．7％、孵化囊胚率为6．7％,显著高于成纤维细胞组成的重构胚（P＜0．05）。我们研究了卵母细胞的采集方法,激活方法和卵龄对卵丘细胞核移植重构胚体外发育的影响。以血清饥饿法将卵丘细胞诱导至G0或G1期,抽吸法／解剖法采集卵母细胞,体外培养33或44h,将卵丘细胞置于去核卵母细胞的卵周隙中,重构胚以钙离子载体A23817或电泳冲结合6－DMAP激活处理,体外培养6天,结果表明,卵 母细胞采集方法、激活液中细胞松弛素（CB）并不影响重构胚的发育（以卵龄44h的卵母细胞为受体）;而以电脉冲结合6－DMAP激活处理能提高重构胚发育能力（以卵龄33h的卵母细胞为受体）（P＜0．05）。本研究显示,以电脉冲结合6－DMAP激活卵丘细胞重构胚,能在体外发育至囊胚。     

第二极体排出时间早晚与胚胎发育潜能相关性的研究

目的探讨第二极体排出时间早晚与胚胎质量及发育潜能之间的关系。方法以本生殖医学中心2009年6月-8月IVF—ET周期患者受精卵子为研究对象,共计1170枚卵子。以受精5h为时间界限将受精胚胎分为第二极体正常排出组（正常组）和延迟排出组（延迟组）。分别观察两组卵子正常／异常受精率（2PN率,1PN和3PN率）和优质胚胎率;同时统计阳性妊娠结局所移植胚胎中,正常组和延迟组的胚胎比例各占多少。采用卡方检验对数据进行统计学处理。结果①两组正常受精卵数目之间以及异常受精卵数目之间均有显著性差异（P〈0．05）;两组总受精卵数目之间有非常显著差异（P〈0．001）。②两组的优质胚胎率之间显著性差异（P〈0．05）。③统计阳性妊娠结局所移植的97个胚胎中,来自于正常组的胚胎有92个（94．9％）,仅5个是来自于延迟组（5．1％）。结论受精5h内排出第二极体的卵子其总受精率、正常受精率以及所发育的胚胎质量均显著高于第二极体出现晚的卵子,而且有着较高的胚胎植入率。对受精5h的卵子进行第二极体观察有助于早期预测患者本次IVF-ET周期胚胎的发育潜能以及妊娠结局;还可以作为决定是否行早补救ICSI的判定指标之一。     

耳聋左慈丸对水杨酸耳鸣模型大鼠听中枢神经元放电的影响

目的：在慢性水杨酸（SA）耳鸣模型上,观察耳聋左慈丸对大鼠下丘外侧核（ICx）和次听皮层（AⅡ）放电的影响,探讨耳聋左慈丸防治耳鸣的神经机制。方法：健康SD大鼠30只,随机平均分成三组：正常对照组、慢性SA耳鸣模型组和耳聋左慈丸防治组。采用立体定位技术及细胞外记录方法,观察不同组别大鼠ICx和AⅡ自发放电活动,用平均自发放电率和放电间隔直方图为观察指标。结果：①慢性SA耳呜模型组与正常对照组大鼠相比,ICx神经单元平均自发放电率增高（4．57±0．54Hz vs 3．14±0．40Hz,P〈0．05）,进一步从放电间隔直方图来分析,短间隔自发放电脉冲数占总放电数比例较高（0-40ms为58％ vs 40％;0～4ms为9％vs5％）。慢性SA耳鸣模型组AⅡ神经单元平均自发放电率与正常对照组大鼠相比有增高趋势（3．844±0．36Hzvs3．17±0．34Hz）,短间隔自发放电脉冲数占总放电数比例较正常对照组增高（0-22ms：31％VS 16％;0—8ms：19％vs16％）。②耳聋左慈丸防治组大鼠与慢性SA耳鸣模型组相比,ICx和AⅡ神经单元平均自发放电率显著降低（ICx：2．41±0．21Hzvs4．57±0．54Hz,P〈0．01;AⅡ：2．24±0．24Hz vs 4．57±0．54Hz,P〈0．01）,短间隔自发放电脉冲数占总放电数比例较低（ICx 0～40 ms：50％vs58％;0-4ms：4％vs9％;AⅡ0～22ms：24％VS 31％;0—8 ms;11％ vs 19％）。结论：慢性SA耳鸣模型动物ICx和AⅡ神经元自发放电活动增加,短间隔放电脉冲数比例较正常对照组增加,耳聋左慈丸能减弱这种变化。     

不同卵龄小鼠卵母细胞激活和受精的比较研究

本实验比较了不同卵龄的小鼠卵母细胞受酒精人工刺激后的激活率和体外受精率,以探索卵母细胞激活和受精的机制。向NIH雌鼠腹腔注射孕马血清促性腺激素（PMSG）7．5单位,48小时后注射人绒毛膜促性激素（HCG）7．5单位,于不同时间杀小鼠,取卵母细胞与卵丘细胞的复合体（OCC）。从注射HCG后到取OCC的时间视为卵母细胞的卵龄。将OCC置于含8％酒精的M2中7分钟,再在16中培养5小时后,用0．3mg/mL的透明质酸酶去卵丘细胞。卵母细胞形成原核或速即卵裂为激活的标志,将OCC加入已获能的精子悬液中,5小时后将从卵丘细胞中释放出来的卵母细胞转移到M16中,将日发生卵裂为卵母细胞体外受精和激活的标志。小鼠卵母细胞卵龄为20h,其激活率为81．6％,速即卵裂率为48．0％;而卵龄进一步增加到24h,激活率和卵裂率转为下降（Table1）。而卵母细胞受精子激活和受精则不同,卵龄为15h ,卵母细胞的体外受精率为45．4％;随着卵龄的进一步增加,体外受精率则下降（Table2)。Fig.1显示：新排出的卵母细胞容易被精子激活而受精;卵龄较大的卵母细胞较易被酒精的人工刺激而激活。可能是卵母细胞从成熟到老化过程中,细胞的结构、功能及对外界刺激的敏感状态都在发生一些规律性的变化,而激活和受精的机制不完全,还不能精对卵龄的要求要严格。     

不同激素和注射方式对家猫超排效果的比较

比较了PMSG／hCG和FSH／hCG两种方案以及PMSG的不同剂量和注射方式对家猫的超排效果的影响。用100IU的PMSG超排家猫所得到的排卵点数及平均每只猫获得的卵数显著低于200IU处理组或300IU处理组（P＜0．05）,但200IU处理组与300IU处理组之间的超排效果也无显著差异（P＞0．05）;用皮下注射200IU的PMSG或用肌肉注射200IU的PMSG对超排效果无差异（P＞0．05）;用200IU PMSG／200IU hCG和1.5mg FSH/200IU hCG两种方案对家猫超排,发现不论是每只猫的排卵点数、卵子获得数,还是卵子的第一极体排放率都没有显著差异（P＞0．05）。实验说明,PMSG的注射方式不影响对家猫的超排效果,用200IU的PMSG超排家猫是较适合的剂量,FSH和PMSG都可用于家猫的超排,但PMSG使用更为方便。     

两种冷冻保护剂对小鼠2-细胞胚冷冻效果的比较

乙二醇（ETG）和1,2-丙二醇（PROH）具有高细胞渗透性和低毒性特点,常被用于人及多种哺乳动物早期胚胎冷冻保存。为了比较ETG和PROH对小鼠2-细胞胚的冷冻保护效果,本试验分别采用这两种冷冻保护剂,对小鼠2-细胞胚进行冷冻保存,并采用冻后体外培养和囊胚移植进行冷冻效果检测。结果表明,PROH组胚胎解冻后胚胎存活率与ETG组无显著差异,但PROH组4-细胞胚发育率和囊胚发育率显著高于ETG组（82．7％vs．64．6％,61．2％vs．29．1％,P〈0．01）。囊胚移植结果表明,2-细胞胚胎冻存后能够发育为正常的后代,PROH组和ETG组的囊胚移植后妊娠产仔率无统计学差异（P〉0．05）,但均显著低于对照组（P〈0．05）。为了分析两组胚胎冻存后损伤情况,埘解冻后的胚胎细胞微丝进行检测,结果显示ETG组微丝受损的胚胎数高于PROH组。本研究结果证明采用PROH作为冷冻保护剂冷冻保存小鼠2-细胞胚的冻存效果优于ETG[动物学报54（6）：1098—1105,2008]。     

Full-term development of golden hamster oocytes following intracytoplasmic sperm head injection

The golden hamster is the mammalian species in which intracytoplasmic sperm injection (ICSI) was first tried to produce fertilized oocytes. Thus far, however, there are no reports of full-term development of hamster oocytes fertilized by ICSI. Here we report the birth of hamster offspring following ICSI. Keys to success were 1) performing ICSI in a dark room with a small incandescent lamp and manipulating both oocytes and fertilized eggs under a microscope with a red light source and 2) injecting sperm heads without acrosomes. All oocytes injected with acrosome-intact sperm heads died within 3 h after injection, while those oocytes injected with acrosomeless sperm heads survived injection. Under illumination with red light in a dark room, the majority of the oocytes injected with acrosomeless sperm heads were fertilized normally (77%), cleaved (91%), and developed into morulae (49%). Of the 47 morulae transferred to five recipient females, nine (19%) developed to live offspring.     

Intracytoplasmic sperm injection of bovine oocytes with stallion spermatozoa

Five experiments were designed to study the fertilizability and development of bovine oocytes fertilized by intracytoplasmic sperm injection (ICSI) with stallion spermatozoa. Experiment 1 determined the time required for pronuclear formation after ICSI. Equine sperm head decondensation began 3 h after ICSI; 42% were decondensed 6 h after ICSI. Male pronuclei (MPN) began to form 12 h after ICSI. Female pronuclei (FPN), however, formed as early as 6 h after ICSI. In Experiment 2, ionomycin, ionomycin plus 6-dimethylaminopurine (DMAP), and thimerosal were used to activate ICSI ova. None of the ICSI ova cleaved after treatment with thimerosal. Ionomycin activation after 24 and 30 h of oocyte maturation resulted in 29 and 48% cleavage rates, respectively. Ionomycin combined with DMAP resulted in 49, 6 and 3% cleavage, morula and blastocyst rates, respectively, when oocytes were activated after 24 h maturation. In Experiment 3, rates of cleavage (45-60%) and development to morulae (4-13%) and blastocysts (1-5%) stages following ICSI were not different (P>0.05) among three stallions. Treatment of stallion spermatozoa with ionomycin did not affect cleavage or development of ova fertilized by ICSI. The chromosomal constitution of blastocysts derived from ICSI was bovine, not bovine and equine hybrids. In Experiment 4, to make male and FPN form synchronously, colchicine and DMAP were used for 4 h to inhibit oocytes at metaphase during activation; 63% of oocytes were still at metaphase 8h after ICSI when treated with colchicine, and 50% of sperm nuclei were decondensed. About 18 h after ICSI, 21 and 50% male and FPN had formed, respectively, but cleavage rates were low, and only 1% developed to morulae. In Experiment 5, to test if capacitated equine sperm could fuse with the bovine oolemma, capacitated spermatozoa were injected subzonally (SUZI). Of the 182 SUZI oocytes, 49 (27%) contained extruded second polar bodies. After activation of oocytes with second polar bodies, 44, 22 and 15% developed to 2-, 4- and 8-cell stages, respectively, but development stopped at the 8-cell stage. None of the unactivated oocytes cleaved. In conclusion, equine spermatozoa can decondense and form MPN in bovine oocytes after ICSI, but subsequent embryonic development is parthenogenetic with only bovine chromosomes being found.     

Offspring from intracytoplasmic sperm injection of aged mouse oocytes treated with caffeine or MG132

Postovulatory mammalian oocytes age significantly in culture. B6D2F1 or ICR strain mouse oocytes were collected 16 h after hCG injection and then cultured for up to 40 h post hCG at 37 °C under 5% CO(2) in air. After intracytoplasmic sperm injection (ICSI), B6D2F1 and ICR oocytes lost full-term developmental potential by 30 h and 26 h after hCG administration, respectively. However, using supplementation with 10 mM caffeine or 1-5 μM of MG132, we could obtain live offspring from oocytes at 34 h (BDF1, 5%-21%) or 28 h (ICR, 5%-18%), whereas none were obtained from untreated aged oocytes. Caffeine maintained normal meiotic spindle morphology, whereas MG132 maintained maturation-promoting factor activity. These treatments did not affect the potential of fresh oocytes for fertilization and subsequent development. Thus, it should be safe to use these chemicals in routine in vitro fertilization and offspring could be generated by ICSI of aged fertilization failed oocytes.     

Intracytoplasmic injection (ICSI) of in vivo or in vitro matured oocytes with fresh ejaculated or frozen-thawed epididymal spermatozoa and additional calcium-ionophore activation in the pig

In Experiment 1, we performed intracytoplasmic sperm injection (ICSI) of frozen-thawed epididymal and fresh ejaculated in vitro-capacitated spermatozoa into in vivo and in vitro-matured porcine oocytes. Within each group, oocytes were sperm-injected, sham-injected or served as handling controls. After subsequent in vitro-culture for 48 h the number of unchanged, fragmented und cleaved oocytes was recorded. The best result (14% cleaved after ICSI vs 2 and 0% with the sham injection and handling controls; P < 0.01) was achieved with fresh in vitro-capacitated spermatozoa injected into in vivo-matured oocytes. In vitro-matured oocytes displayed high fragmentation rates. In Experiment 2, in vitro matured oocytes were injected with freshly ejaculated in vitro-capacitated spermatozoa, followed by a 5 min-exposure to 0 (control), 50 or 100 microM calcium-ionophore. Comparable groups were sham injected or served as handling controls. It became apparent that Ca-ionophore treatment after injection of spermatozoa was ineffective at 100 microM, where at 50 microM a significant reduction in cleavage rate was observed (6 vs 26% with untreated controls, P < 0.01). Fluorescence staining with Hoechst 33342 revealed that in most cases of sperm-injected oocytes that remained unchanged after 48 h of in vitro-culture, sperm heads had not decondensed. Only few oocytes had continued to the pronucleus stage. In this context no favorable effect of Ca-ionophore was to be observed.     

Development of in vivo-matured porcine oocytes following intracytoplasmic sperm injection

The objective of this study was to assess the development of porcine ova fertilized by intracytoplasmic sperm injection (ICSI). Allyl trenbolone (Regumate) was used to synchronize estrus in 13 postpuberal gilts. Gilts were superovulated with pregnant mare serum gonadotropin and hCG. Ova were aspirated from 5- to 8-mm follicles at 36 h after hCG. Cumulus cells were removed by blunt dissection and pipetting in Beltsville embryo culture medium (BECM) supplemented with 0.1% hyaluronidase. Sperm were washed and resuspended in BECM + 8% polyvinylpyrrolidone. Ova (n = 237) that exhibited a polar body were centrifuged at 15 000 x g for 6 min and injected with a single spermatozoon. One hundred fifty-four ova were cultured in NCSU-23 medium in a 5% CO(2) in air environment for 168 h. Ova were fixed in acetic acid/ethanol and stained with 1% orcein. Sixty-nine ICSI ova were cultured for 24 h and transferred (mean = 23) to three recipients. Eighty-one ova (69%) that survived ICSI cleaved within 48 h. Thirty-eight percent (31/81) of these ova became blastocysts (mean +/- SEM = 24.7 +/- 1.1 cells). One recipient gave birth to three pigs. These results demonstrate that porcine embryos derived from ICSI can develop into live pigs.     

An attempt at intracytoplasmic sperm injection of frozen-thawed minke whale (Balaenoptera bonaerensis) oocytes.

Little is known about the characteristics of fertilisation events in minke whales. Cryopreserved minke whale oocytes and spermatozoa do not fertilise in a standard IVF. This study was conducted to investigate the pronucleus formation ability of cryopreserved minke whale oocytes and their subsequent development following intracytoplasmic sperm injection (ICSI). In experiment 1, frozen-thawed minke whale immature oocytes were cultured for in vitro maturation (IVM) in a maturation medium (TCM199) supplemented with either porcine follicle stimulating hormone (pFSH)/estradiol-17beta (E2) or pregnant mare's serum gonadotropin (PMSG)/human chorionic gonadotropin (hCG). After 120 h of IVM, oocyte survival was examined before ICSI, and showed no significant difference in morphological normality (24-36%) between the two IVM media. Two-cell embryos (two oocytes from 21 sperm-injected oocytes) were obtained when the maturation medium was supplemented with pFSH/E2 or PMSG/hCG. In experiment 2, cryopreserved maturing oocytes were investigated for the effects of repeat-culture (2 h or 24 h) on survival before ICSI. Pronuclear formation and development were examined for the effects of sperm pretreatment with dithiothreitol (DTT) and oocyte activation with ethanol at ICSI. A frequency of 49-69% of frozen-thawed maturing oocytes was used for ICSI. Although oocyte activation did not produce a significant difference in survival, pronucleus formation and embryonic development, 2- and 4-cell cleaved oocytes were observed after injection of sperm pretreated with DTT.     

In vitro fertilization and embryo development in vitro and in vivo in the tiger (Panthera tigris)

A study was conducted to evaluate the adaptability to the tiger of an in vitro fertilization/embryo culture system previously developed in the domestic cat. In Trial I (July 1989), 10 female tigers were treated with either 2,500 (n = 5) or 5,000 (n = 5) IU eCG i.m. and with 2,000 IU hCG i.m. 84 h later. In Trial II (January 1990), 6 females (5 of which were treated in Trial I) were given 2,500 IU eCG i.m. and 2,000 IU hCG i.m. 84 h later. Twenty-four to twenty-six hours after hCG treatment, all tigers were subjected to laparoscopy, and oocytes were aspirated transabdominally. On the basis of follicular development (follicles greater than or equal to 2 mm in diameter), all females responded to exogenous gonadotropins (range, 6-52 follicles/female). Follicle number and oocyte recovery rate were unaffected (p greater than 0.05) by eCG dose or time of year. A total of 456 oocytes were collected from 468 follicles (97.4% recovery; mean, 28.5 +/- 3.4 oocytes/female). Of these, 378 (82.9%) qualified as mature, 48 (10.5%) as immature, and 30 (6.6%) as degenerate. During Trial I, 8 electroejaculates were collected from 7 male tigers, and in Trial II, 3 semen samples were collected from 3 males. Motile sperm were recovered on each occasion; the overall mean (+/- SEM) ejaculate volume was 7.5 +/- 0.7 ml, the number of motile sperm/ejaculate was 105.9 +/- 20.6 x 10(6), and the percentage of structurally normal sperm/ejaculate was 81.4 +/- 2.0%. After swim-up processing, 0.05 x 10(6) motile sperm were co-cultured with 10 or fewer tiger oocytes in a humidified atmosphere (38 degrees C) of 5% CO2 in air. Of the 358 mature oocytes inseminated, 227 (63.4%) were fertilized. Oocytes from 2 females became contaminated in culture and, therefore, were excluded from embryo cleavage calculations. Of the remaining 195 fertilized oocytes, 187 (95.9%) cleaved to the two-cell stage. No parthenogenetic cleavage was observed in noninseminated control oocytes (n = 20). Eighty-six good-to-excellent-quality two- to four-cell embryos were transferred surgically into the oviducts of 4 of the original oocyte donors in Trial I and 2 females in Trial II. A pregnancy occurred in 1 female in Trial II, and 3 live-born cubs were delivered by Caesarean section 107 days after embryo transfer. Of the 56 cleaved embryos cultured in vitro in Ham's F10 for 72 h, 14 (25.0%) were at the sixteen-cell stage, and 15 (26.8%) were morulae.(ABSTRACT TRUNCATED AT 400 WORDS)     

Viable piglets generated from porcine oocytes matured in vitro and fertilized by intracytoplasmic sperm head injection

Intracytoplasmic sperm injection (ICSI) of a nonmotile cell into the ooplasm for assisted fertilization is a highly specialized procedure for producing the next generation. The production of piglets by ICSI has succeeded when in vivo-matured oocytes have been used as recipients. Our objective was to generate viable piglets by using porcine oocytes matured in vitro and fertilized by ICSI after evaluating the efficacy of using donor spermatozoa in which the acrosome had been artificially removed by treatment with calcium ionophore A23187 (Ca-I). The rate of acrosomal loss in spermatozoa was increased significantly as the duration of treatment with 10 micro M Ca-I was prolonged for 30-120 min (Ca-I treated; 55.6-78.6%), whereas the rate was not different as the duration of incubation without Ca-I was prolonged for 30-120 min (control; 45.3-58.4%). On the sixth day of in vitro culture after injection of the sperm head and subsequent stimulation with an electrical pulse, the rates of blastocyst formation were not significantly different between the two groups: the rates for oocytes injected with Ca-I-treated sperm heads (incubated for 120 min) and for those injected with control sperm heads were 8.6% and 4.0%, respectively. The mean cell numbers of the blastocysts were not significantly different between the two groups (25.6 and 22.7, respectively). Within 2 h after the stimulation, the injected oocytes were transferred to estrous-synchronized recipients. The three recipients that received oocytes injected with Ca-I-treated sperm heads (77-150 oocytes per recipient) were not pregnant, whereas two of the four recipients given oocytes injected with control sperm heads (55-100 oocytes per recipient) were pregnant. One of these farrowed three (a male and two female) healthy piglets. The results demonstrate clearly that in vitro-matured oocytes injected with sperm heads are developmentally competent and can produce viable piglets. They also suggest that removal of the acrosome from the spermatozoon before injection does not affect the development of the blastocyst in vitro. This might not also improve the production of piglets in vivo.     

Prolonging the interval from ovarian hyperstimulation to laparoscopic ovum pick-up improves oocyte yield, quality, and developmental competence in goats

The objective was to evaluate the effect of the interval between ovarian hyperstimulation and laparoscopic ovum pick-up (LOPU) on quality and developmental competence of goat oocytes before and after in vitro maturation (IVM) and intracytoplasmic sperm injection (ICSI). Estrus was synchronized with an intravaginal insert containing 0.3g progesterone (CIDR) for 10d, combined with a luteolytic treatment of 125 microg cloprostenol 36 h prior to CIDR removal. Ovaries were hyperstimulated with 70 mg FSH and 500 IU hCG given im 36, 60, or 72 h prior to LOPU (n=15, 16, and 7 does, respectively). For these groups, oocyte retrieval rates (mean+/-S.E.M.) were 24.7+/-2.9, 54.5+/-4.7, and 82.8+/-4.6% (P<0.001), and the proportions of cumulus-oocyte complexes (COC) with more than five layers of cumulus cells were 29.7+/-8.3, 37.6+/-6.9, and 37.3+/-7.0% (P<0.001). The proportion of IVM oocytes was highest at 72 h (82.1+/-2.8%; P<0.05), with no significant difference between 36 and 60 h (57.3+/-8.9% and 69.0+/-8.4%). Cleavage rates of ICSI embryos were 4.2+/-4.2, 70.9+/-8.4, and 78.9+/-8.2% with LOPU 36, 60, and 72 h post FSH/hCG (P<0.01), with a lower proportion of Grade-A embryos (P<0.05) following LOPU at 36 h compared to 60 and 72 h (29.7+/-8.3%, 37.6+/-6.9%, and 37.3+/-7.0%). In summary, a prolonged interval from FSH/hCG to LOPU improved oocyte retrieval rate and oocyte quality. Therefore, under the present conditions, LOPU 60 or 72 h after FSH/hCG optimized yields of good-quality oocytes for IVM and embryo production in goats.